Stem cell potential relies on their capacity to differentiate in defined cell types and integrate into corresponding tissues and organs. Another profitable feature of stem cells is their paracrine release of cytokines, interleukines, thrphic factors and growth factors.
Current research and clinical trials are being designed to probe the therapeutic effect of stem cells in several pathologies, and there is an increasing demand for stem cell-based therapies.
Certain degenerative diseases of the respiratory system, cardiovascular system, immune system, endocrine system/function, central and peripheral nervous systems, spinal cord injury, ischemia/reperfusion injury and demyelinating diseases have an inflammatory component mediated by reactive oxygen species (ROS) named oxidative stress.
Reactive oxygen species, principally superoxide anion radical (O2−) and its dismutation product H2O2, are natural waste subproducts in mitochondria of cells where respiratory chain takes part, a phenomenon vital for cell life due to its function in energy molecule (ATP) generation.
Mitochondria are the most redox-active compartment of mammalian cells, accounting for more than 90% of electron transfer to O2 as the terminal electron acceptor. The predominant electron transfer occurs through a central redox circuit which uses the potential energy available from oxidation of various metabolic substrates (e.g., pyruvate, fatty acids) to generate ATP. Regulation of this process is central to cell function because cells must produce ATP while at the same time maintain an appropriate homeostasis in terms of supply of non-essential amino acids, eliminate excess amino acids, supply glucose and interconvert energy precursors to allow for long-term energy supply in the face of variable and intermittent food intake. Part of the regulation appears to occur through a continuous low rate of ROS generation and molecular sensors. The associated redox circuitry for this regulation, although poorly defined is known to require a specialized redox environment.
Under excessive oxidative stress, simultaneous collapse of the mitochondrial ATP-generation potential and a transient increase in ROS generation by the electron transfer chain, can result in mitochondrial release of ROS to cytosol. This can trigger “ROS-induced ROS release” in neighboring mitochondria. Thus, although a low rate of ROS generation is a normal process in mitochondria, disruption of electron flow with excessive ROS generation can result in senescence, apoptosis and cell death. Go and Jones, 2008. Redox compartmentalization in eukaryotic cells. Biochimica et Biophysica Acta 1780 (1273-1290); Zorov, Juhaszova and Sollott. 2006. Mitochondrial ROS-induced ROS release: an update and review. Biochim Biophys Acta 1757 (509-517).
Indeed, these processes are directly relevant to mitochondrial oxidative stress-related diseases such as Parkinson's disease, Friedrich's ataxia, Huntington disease and diabetes. Go and Jones, 2008. Redox compartmentalization in eukaryotic cells. Biochimica et Biophysica Acta 1780 (1273-1290); Dringen, Gutterer and Hirrlinger, 2000. Glutathione metabolism in brain. Metabolic interaction between astrocytes and neurons in the defense against reactive oxygen species. Eur J Biochem 267 (4912-4916); Chinta and Andersen, 2008. Redox imbalance in Parkinson's disease. Biochimica et Biophysica Acta 1780 (1362-1367); Cohen, 2000. Oxidative stress, mitochondrial respiration, and Parkinson's disease. Ann N Y Acad Sci 899 (112-120); Lodi, Tonon, Calabrese and Schapira, 2006. Friedreich's ataxia: from disease mechanisms to therapeutic interventions. Antioxid Redox Signal 8 (438-443); McGill and Beal, 2006. PGC-1alpha, a new therapeutic target in Huntington's disease? Cell 127 (465-468); Donath, Ehses, Maedler, Schumann, Ellingsgaard, Eppler and Reinecke, 2005. Mechanisms of beta-cell death in type 2 diabetes. Diabetes 54 (Suppl 2) (S108-S113).
Peroxides, including hydrogen peroxide (H2O2), are one of the main reactive oxygen species (ROS) leading to oxidative stress. H2O2 is continuously generated by several enzymes (including superoxide dismutase, glucose oxidase, and monoamine oxidase) and must be degraded to prevent oxidative damage. The cytotoxic effect of H2O2 is thought to be caused by hydroxyl radicals generated from iron catalyzed reactions, causing subsequent damage to DNA, proteins and membrane lipids. H2O2 acts as a “suicide substrate” at high concentrations (>100 μM), leading to an irreversible inactivation of catalase. Hyslop, Zhang, Pearson y Phebus, 1995. Measurement of striatal H2O2 by microdyalysis following global forebrain ischemia and reperfusion in the rat: Correlation with the cytotoxic potential of H2O2 in vitro. Brain Res 671 (181-186). H2O2 causes intracellular glutathione depletion, a molecule that remove H2O2 from the cell, suggesting that H2O2 enters the cells and therefore may set in motion one or more toxic pathways in cells. Dringen, Pawlowski and Hirrlinger, 2005. Peroxide Detoxification by Brain Cells. J Neurosci Res 79(157-165); Halliwell and Whiteman, 2004. Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? British Journal of Pharmacology 142 (231-255); Baud, Greene, Li, Wang, Volpe and Rosenberg, 2004. Glutathione Peroxidase-Catalase Cooperativity Is Required for Resistance to Hydrogen Peroxide by Mature Rat Oligodendrocytes. J Neurosci 24(1531-1540).
Cells also synthetize antioxidative molecules and have mechanisms for recycling them. Gluthation (GSH) is one of the principal proteins involved in the antioxidant machinery eliminating H2O2, together with its oxidized form GSSG and related enzymes glutathione peroxidase (GPx), glutathione reductase (GR), glutaredoxin and NADPH/NADP+. A variety of studies using cell culture models support the crucial role played by GSH in mitochondria as a protective effect in apoptotic cell death. In apoptosis, programmed cell death, oxidation of mitochondrial GSH/GSSG stimulates GSH depletion resulting in increased ROS, suggesting a role for GSH in controlling mitochondrial ROS generation. Dringen, Pawlowski and Hirrlinger, 2005. Peroxide Detoxification by Brain Cells. J Neurosci Res 79(157-165); Dringen, Gutterer and Hirrlinger, 2000. Glutathione metabolism in brain. Metabolic interaction between astrocytes and neurons in the defense against reactive oxygen species. Eur J Biochem 267 (4912-4916).
Another enzyme in the antioxidant machinery that eliminates hydrogen peroxide is catalase. Catalase is a cytoplasmic enzyme that is of special relevance when the clearance of H2O2 in high concentrations is required. Baud, Greene, Li, Wang, Volpe y Rosenberg, 2004. Glutathione Peroxidase-Catalase Cooperativity Is Required for Resistance to Hydrogen Peroxide by Mature Rat Oligodendrocytes. J Neurosci 24(1531-1540).
It has also been probed that hMSCs possess the main enzymatic and non-enzymatic mechanisms to detoxify reactive species and to correct oxidative damage of proteome and genome that ensure the efficient manage of ROS. Valle-Prieto and Conget, 2010. Human Mesenchymal Stem Cells efficiently manage oxidative stress. Stem Cell Dev 19 (1885-1893). If this potential is maintained in vivo, hMSCs could also contribute to tissue regeneration limiting ROS-induced tissue damage.
Some successful attempts to modify the synthesis of enzymes involved in elimination of ROS describe that human Bone Marrow Stromal Cells cultured in the presence of ascorbate express higher levels of superoxide dismutase, catalase and glutathione (Stolzing and Scutt, 2006. Effect of reduced culture temperature on antioxidant defenses of mesenchymal stem cells. Free Radic Biol Med 41(326-338). Moreover, in the article, Ebert, Ulmer, Zeck, Meissner-Weigl, Schneider, Stopper, Schupp, Kassem and Jacob, 2006. Selenium supplementation restores the antioxidative capacity and prevents cell damage in bone marrow stromal cells in vitro. Stem Cells 24(1226-1235), the authors describe the up-regulation of the basal antioxidant capacity of BMMSCs by modifiying the cell culture conditions with selenium supplementation or temperature reduction. Stolzing and Scutt (2006) published that the temperature reduction in these BMMSC doesn't affect their viability but that increases their differentiation. On the other hand, stem cells directly obtained through the treatment method of the present invention, cells named HC016, don't show any evidence of differentiation, maintaining their undifferentiated phenotype, and also their viability.
Furthermore, Ebert et al., 2006 demonstrate that the selenium supplemetantion of the culture medium of BMMSC with 100 nM sodium selenite exclusively increases the activity of intracellular selenium-dependent enzymes, as glutathione peroxidase (GPx) y la thioredoxin reductase (TrxRs). On the other hand, HC016 cells keep their viability, proliferative capacity and undifferentiated phenotype, and also activate genes coding for key selenium-independent enzymes for ROS detoxification, like superoxide dismutases (SODs) y catalase (Cat), fundamental to ROS detoxification. Moreover, HC016 cells have increased levels of GSH.
To conclude, HC016 cells directly obtained with the treatment method described in the present invention show a series of advantages with respect to the stem cells used in the state-of-the-art, that renders HC016 cells specially suited to act in oxidative stress conditions. These advantages are mainly 1) generation of a superior intracelular pool of the detoxifying molecule GSH, 2) a superior and increased expression of genes coding for enzymes involved in reactive oxygen species elimination, 3) a new cytoskeletal conformation and therefore, an consequently, a higher migration capacity towards damaged areas, and 4) a higher expression of growth factors related to tissue regeneration processes.
These effects acquired by HC016 cells, increase their intracelular and extracelular defenses against ROS, without generating any modification regarding their viability and differentiation state.
WO 2010/150094 describes a method for mesenchymal stem cells in vitro differentiation into adipocytes and its use as a cell therapy. The method described consists in culturing those cells in hypoxic conditions.
WO2007/030870 provides a method for stem cell differentiation, more precisely, cells from human embryos (hES cells), into cardiomyocites and neural progenitors by culturing hES cells in a medium without serum, that additionally contains prostaglandin or a p38MAP Kinase inhibiting molecule.
As a consequence, there is an important need in the state-of-the-art to generate methods for obtaining mesenchymal stem cells with improved or increased own enzymatic and non-enzymatic mechanisms focused on the elimination of reactive oxygen species, and as a consequence, generate cells that could be more effectively used in cell therapies for oxidative stress associated diseases.